Project Summary/Abstract Our goal is to elucidate autophagy-lysosomal mechanism in the pathogenesis of Parkinson's disease (PD). Early pathological analysis of PD and recent studies of PD-linked genes, such as SNCA, LRRK2, Parkin, Pink1, GBA and ATP13A2, implicates dysfunctional autophagy-lysosomal pathway in the pathogenesis of PD. Recent evidence shows that inhibition of LRRK2 or expression of LRRK2 PD mutants causes aberrant autophagic activity, but the detailed mechanism is unclear. Our preliminary study suggests a link of LRRK2 to an autophagy kinase, which is essential for autophagy and required for axon/neurite outgrowth. Here we propose to investigate the molecular mechanism whereby LRRK2 regulates autophagy through directly targeting autophagy machinery and dysfunctional autophagy as a potential PD pathogenic pathway. We previously reported that disruption of autophagy in mice leads to age-dependent accumulation of endogenous ?-Syn in dystrophic axons and altered dopamine transmission, consist with impaired autophagy as one of the failing cellular mechanisms involved in the pathogenesis of PD. Emerging evidence suggests that autophagy not only participates in degradation of ?-Syn, but also is involved in the secretion pathway of ?-Syn, which underlies the spreading of synucleiopathy. Fibrillar ?-Syn can be secreted through exosomes and/or autophagosome-related structures. Furthermore, increasing autophagy gene beclin1 or transcription factor TFEB expression facilitates the autophagic clearance of ?-Syn and offers neuroprotection in rodent models. However, the precise mechanism for the autophagic clearance of intracellular ?-Syn remains poorly characterized. Finally, whether LRRK2 also contributes to the control of ?-Syn homeostasis through regulation of autophagy is unclear. Given the emerging role of autophagy in the exocytosis of ?-Syn, we hypothesize that autophagy regulates ?-Syn clearance through both degradative and secretory pathways; LRRK2 modulates ?- Syn homeostasis by targeting autophagy. In Project 3 we propose (1) to determine molecular and genetic basis for LRRK2-mediated autophagy regulation. We will use biochemical, cellular and animal models to dissect the role for LRRK2 wildtype and PD mutation G2019S in autophagy control through modulating the autophagy kinase and SNARE-related protein trafficking; (2) to determine the mechanism that autophagy and LRRK2 modulate ?-Syn homeostasis by targeting multiple trafficking pathways. We will apply multidisciplinary approaches to test that LRRK2 and autophagy pathway converge to regulate ?-Syn homeostasis by targeting secretion and degradation in cell and animal models of PD. Our project 3 will be conducted in close collaboration with Drs. Jie Shen and Tom Sdhof, who have extensive experience in LRRK2 and ?-Syn research, respectively. Completion of our project 3 is expected to provide timely knowledge for understanding LRRK2 pathogenic mechanism and developing potential therapeutic strategies by targeting autophagy-lysosome pathways for the clearance of ?-synuclein.